Process for preparing high molecular weight products of apathogenic anaerobic cultures



United States Patent 3,303,096 PROCESS FOR PREPARING HIGH MOLECULARWEIGHT PRODUCTS 0F APATHOGENIC AN- AEROBIC CULTURES Heinz Feier,Lorsbach, Taunus, Dietmar Gericke, Frankfurt am Main, Georg Nesemann,Lorsbach, Taunus, and Paul Priive, Hofheim, Taunus, Germany, assignorsto Farbwerke Hoechst Aktiengesellschaft vormals Meister Lucius 8rBruning, Frankfurt am Main, Germany, a corporation of Germany N0Drawing. Filed Sept. 21, 1964, Ser. No. 398,088 Claims priority,application Germany, Sept. 25, 1963,

40,829 12 Claims. (Cl. 167-78) It is known that from gram-negativeaerobic bacteria and from the filtrates of their cultures substances canbe isolated which increase the unspecific resistance of the animal andhuman organism.

These substances are high molecular weight lipopolysaccharides which arestrongly pyrogenic and toxic. in the administration of these compounds,one can observe that a negative phase with a reduction of the resistanceprecedes the increase of the resistance, which renders the productsunsuitable for pharmaceutical purposes.

It has hitherto not been possible to isolate substances that have anaction on the unspecific resistance from gram-positive aerobic bacteria.of gram-positive aerobes, only substances which promote the emigrationof leukocytes and which are effective in inflammations caused bygram-positive aerobic bacteria have been found.

Now, we have found that high molecular weight substances which increasethe resistance of the organism without previously reducing it and whichdo not increase body temperature can be isolated from culture filtratesof the cultures of apathogenic anaerobic bacteria. It was surprisingthat these substances obtained from culture filtrates of apathogenicanaerobic bacteria do not possess the disadvantageous effects of thelipopolysaccharides obtained from gram-negative bacteria.

As apathogenic, anaerobic bacteria which may be used in the process ofthe present invention, there may be used in the first instanceclostridia. Among these, there may be mentioned, by way of example,Clostridium butyricum (ATCC No. 13 732), Clostridium pectinovemm (NB1386), Clostridium tyrobutyricum (McClung 1750), and Clostrz'diumacetobutylicum (McClung 632).

The above-mentioned bacteria are cultivated in the manner usual foranaerobes, for example in standing flasks or in fermenters such as thoseused for the preparation of antibiotics. As nutrient media, the usualnutrient media for anaerobes which contain either a reducing agent suchas ascorbic acid or iron nails are used, or such nutrient media in whichthe oxygen has been replaced by an inert gas, for example those throughwhich nitrogen is passed.

The culture is allowed to grow for about 3 to 21 days, the culture mediabeing kept at a temperature in the range of 20 and 40 C., preferably atabout 37 C. As a rule, the germs have formed spores after this period.

The bacteria are separated in known manner, for example bycentrifugation or filtration, from these anaerobe cultures, so thatsterile culture filtrates are obtained. Further isolation andpurification of the active products contained in these culture filtratescan be carried out by dialysis and/or by precipitation with solvents.

In dialysis, the active products remain in the inner dialysate sincethey are not dialyzable.

A further purification, which often also brings about an increase inactivity, can be effected by precipitation of the accompanyingsubstances or by precipitation of In the culture filtrates' 3,303,096Patented Feb. 7, 1967 the active products themselves by means ofsuitable solvents.

If water-miscible lower aliphatic alcohols, for example, methanol,ethanol or propanol, are used as solvents, the active substances remainin solution and can be separated with the solution from the precipitatedaccompanying substances. The purification with alcohols is preferablycarried out by adding a twofold or tenfold, preferably a five-fold,quantity of alcohol to the culture filtrate or to its dialysate, which,if desired, may previously be concentrated. The precipitate is thenseparated and discarded and the liquid phase is concentrated to isolatethe active substance. It is advantageous to carry out this operationunder reduced pressure.

If water-miscible ketones such as acetones or methylethyl ketone areused as solvents, the active products are precipitated and theaccompanying substances remain in solution. In this case it isadvantageous to add a considerable excess of ketone, for example anexcess of about one hundredfold the volume of the culture filtrate orits dialysate. The latter is preferably concentrated beforehand. Theprecipitated solids are separated, dried if desired or required, andredissolved in water if desired.

By successive application of the above-described purification methods,it is possible to purify the active substances to a large extent.

The high molecular weight substances obtained by the process of thepresent invention are distinguished by the fact that they increase theresistance, in particular the resistance to infections, without being.pyrogenic or toxic or causing a reduction of the bactericidal potencyof the serum. In addition, repeated administration of the substancesdoes not cause sensitization of the organism treated. TheShwartzmann-Sanarelli phenomenon, which always appears upon applicationof the lipopolysaccharides from gram negative spores mentioned in theintroductory part of this specification, does not appear afterapplication of the products of the invention. An anaphylactic shock islikewise not observed.

The following tests, the results of which are disclosed in thesubsequently following examples, are suitable for characterizing thesubstances:

(1) Test for bactericidal potency of serum (modified according to H.Franck).For determining the bactericidal potency of the serum, mice thathave a weight of 20 g. are intraperitoneally (i.p.) injected with thetest substance. Blood is taken prior to the injection and atpredetermined times, about 4, 16 and 24 hours, after the injection fromthe retro-orbital plexuses. The serum obtained from the blood taken from6 mice is combined to a serum pool, diluted with a physiological saltsolution in a ratio of 1:10 and divided into two equal parts. One partis inactivated at C., whereas the other part remains active. To bothsamples is then added a quantity, corresponding to half the volume ofthe samples, of a suspension of Bacillus subtilis spores which' havebeen cultivated synchronously and thus constitute a so-calledsynchronous culture, i.e., a culture which has in each unit of volumethe same number of cells (for example, 8 10 spores per ml.). Thus, oneactive and one inactive sample of serum are then available for measuringthe growth of B. subtilis spores in the presence of serum.

The growth of the Bacillus subtilis spores in the untreated and in thetreated serum is then determined manometrically by measurement of theoxygen consumption with a Warbu'rg apparatus. 2.0 ml. of serumsubtilissuspension are introduced by means of a pipette into the main space ofeach vessel of this apparatus and 0.1 ml. of a 10% potassium hydroxidesolution are introduced into the central part of each vessel. The

oxygen consumption is measured at intervals of 30 minutes and at 37 C.,an amplitude of 3 cm. and a frequency of 50 rev./min. The decrease ofthe pressure in the vessel is plotted linearly against the time of thetest and gives thus the breathing curves of the subtilis spores. If thetest substance causes an increase of the bactericidal potency of theserum, the oxygen consumption rises rapidly, first in the inactivatedsample (INA) and after a certain period of time, in the active sample(A). The difference in time between these increases in the oxygenconsumption, expressed in minutes, represents the inhibition of thegrowth due to increased bactericidal action of the serum. A quotient, Qis formed between the difference (INAA) for a control sample and thedifference (INAA) fora test sample:

( )control 1 )test sample t QSB The control sample is prepared fromblood taken prior to injection of the test substance and permitsmeasurement of the normal bactericidal potency of the serum. The testsample is prepared ctrom blood taken after administration of the testsample. The greater the bactericidal action of the substance tested, thesmaller is the values of Q i.e., the resistance of the serum per se hasbeen increased by the presence of resistanceincreasing substances.Inactive substances show a value of Q -1.0, and Q 1 means that the testsubstance reduces the bactericidal .potency of the serum.

(2) Infection test-Each animal of groups of 10 to 20 mice (weight ofeach animal about 20 g.) is injected intraperitoneally with 1X5 or 10gamma of test substance. Control animals are given the same amount ofphysiological salt solution. 24 hours after injection of the testsubstance, a hemolyzing Staphylococcus strain is injected intravenouslyinto the animals for infection in such a dose that about 50% of thecontrol animals die.-

The number of surviving animals is determined on each of thesubsequently following 17 days.

The following examples illustrate the invention but they are notintended to limit it thereto:

EXAMPLE 1 (a) Cultivation of anaerobic apatlzogenic Clostridium straina-Prestage cltlture.An Erlenmeyer flask was charged with 150 ml. of anutrient solution consisting of Percent Meat peptone 22 Yeast extract0.5 Meat extract 0.25 Sodium chloride 0.25 Iron nails 1 Tap water(pH-value 7.2).

The solution was innoculated with lyophilized spores of Clostridiambutyricam from an ampule and then covered with sterile paraflin oil.After incubation for 24 hours, at 37 C., the solution showed a goodgrowth of spores.

,B-Mainstage culture.'Erlen;meyer flasks having a capacity of 2 literswere charged with 1.5 l. of a nutrient solution consisting of PercentCasein peptone 0.5 Soya peptone 0.3 Cane sugar 0.5 L-lysine 0.05 NaCl0.5 See. potassium phosphate 0.25 Iron nails (in bags) 1 and sterilizedfor 20 minutes at 121 C. in an autoclave. Immediately after cooling,each of the solutions was inoculated with 50 ml. of the above prestageculture 4 which was withdrawn by means of a pipette below the paraflinoil. After incubation for 12 days at 37 C., 72% of the bacteria hadformed spores, 5% showed beginning spore formation and the restconsisted of rods.

5 I (b) Recovery of the active substances 2 liters of Clostridia culturewere clarified in a centrifuge (system Cepa Sharples) and then filteredwith suction through a degerminating layer. The filtrate was coveredwithtoluene in order to prevent growth of bacteria and then dialyzed ina dialysis bag for 24 to 72 hours against flowing distilled water.Thesolution was then freeze-dried. Yield: g.

4 g. of the product were dissolved in 30 cc. of water and 150 cc. ofethyl alcohol were added while'stirring. The substance which hadprecipitated was again dissolved in 30 cc. of water and 150 cc. of ethylalcohol were added while stirring. The precipitated substance showed noactivity. The combined alcohol phases were concentrated under reducedpressure and gave a brown oil. Yield 2.04

(c) Test for the bactericidal potency of the serum (d) Infection test 10mice were given each 10 gamma i.p. o fthe substance obtained accordingto (b) in 0.4 cc. of water and 24 hours later infected by intravenousinjection of Staphylococcus aureus. Control animals were given the samequantity of physiological salt solution. The following table shows thesurvival rate of the test animals as a function of time:

F TABLE 1 [Survival rate of mice in the infection test] Number ofsurviving animals treated Untreated Days after with 10 gamma of controlinfection the substance obanimals tained according to (b) 0 10 10 l 9 82 6 6 4 9 0 5 8 5 0 7 3 7 7 2 8 7 2 9 7 2 1O 7 2 0 11 7 2 12 6 2 l3 6 214 (i 2 l5 6 2 1G 6 2 17 6 1 The table shows that the death rate of theanimals treated with the preparation of the invention is lower than thatof the untreated control animals, i.e. that the survival rate has beenincreased.

(e) Pyrogenicity pyrogenic in rabbits at -50 gamma/ kg. s.c.

5 r Toxicity A toxicity of the preparation could not be found. The LD inmice is higher than 2 g./ kg.

EXAMPLE 2 (a) Cultivation of an apathogenic Clostridium straina-Prestage cuIture.Erlenmeyer flasks having a capacity of 300 cc. werecharged with 150 ml. of a nutrient solution consisting of Percent Starch0.5 Casein peptone 2 Meat extract 0.5 Yeast extract 0.5

NaCl 0.25

and then sterilized. The solutions were then inoculated with 5 ml. of aniron nail culture of Clostridium bittyricum which showed good sporeformation. The flasks were then placed in an exsiccator which wasevacuated and then filled with nitrogen. The tightly closed exsiccatorwas then stored in an incubator at 37 C. After 12- 18 hours, thenitrient solution was densely overgrown with vegetative rods. Thesesolutions served as the inoculation material for the mainstage culture.

B-Mainstage culture.Ferrnenters provided with an air inlet, a samplewithdrawal device and an inoculation nipple, were charged with 15 litersof a nutrient solution consisting of Percent Cane sugar 0.5 Caseinpeptone 2 Meat extract 0.1 L-lysine 0.05 KCl 0.25 FeSO 0.006

Distilled water.

having a pH-value in the range of 7.6-7.8, and sterilized. The solutionwas then inoculated with 150 ml. of the above prestage culture andincubated at 37 C., while slowly passing nitrogen therethrough. After 10days, 85% of the strain had formed spores.

(b) Recovery of the active substances 12 liters of Clostridia culturewere centrifuged and completely clarified by filtration. After dialysisof this culture filtrate for 24-72 hours under toluene, the solution wasconcentrated to 300 cc. under reduced pressure and 1.5 l. of ethanolwere added with stirring.

The precipitate was taken up in 150 cc. of water and again combined,while stirring, with 750 cc. of ethanol and then centrifuged. Theprecipitate showed a weak action. Both ethanol phases were combined andconcentrated under reduced pressure to 150 cc. 15 liters of acetone wereadded, while stirring; the whole was then centrifuged. The precipitateseparated by centrifugation was washed with ether and dried in anexsiccator. Yield: 3.91 g.

() Test for the bactericidal potency of the serum Upon application ofgamma of the active substance, the Q -value was 0.3 after 16 hours, andupon application of 1 gamma the Q -value was likewise 0.3. After 4hours, the respective values were 0.3 and 0.6. For comparison, theactivity of a lipopolysaccharide from Escherichitl coli, which uponapplication of a dose of 3 gamma gave a Q -value of 1.2, was alsomeasured.

(d) Infection test mice were treated intraperitoneally with 5 gamma ofthe substance obtained according to (b) in 0.5 cc. of water, 24 hoursprior to intravenous infection with Staphlococcus aureus. Controlanimals were given the same amount of physiologic saltsolution. Thefollowing table shows the survival rate of the animals.

TABLE 2 Number of surviving animals treated Untreated Days after with 6gamma 0! control infection the substance obanimals mined according to(b) O 10 1O 1 10 10 2 S 7 3 8 6 4 8 4 5 7 3 G 7 3 7 7 2 8 6 l 9 6 1 10 51 l1 5 l 12 5 l 13 5 1 l4 5 1 15 4 1 1G 3 1 17 3 1 (e) Pyrogenicity Thepreparation obtained according to (b) is nonpyrogenic in rabbits at 100and 200 gamma/kg. i.v.

(f) Toxicity A toxicity of the preparation could not be found. The LD inmice is above 10 g./kg.

EXAMPLE 3 (a) Cultivation of an apathogenic Clostridium straina-Prestage culture.Erlenmeyer flasks having 21 ca pacity of 500 cc. werefilled with 300 cc. of a nutrient solution consisting of Percent Canesugar 0.5 Casein peptone 2 Meat extract 0.1

L-lysine 0.05 NaCl 0.25 pH 7.0.

and sterilized in an autoclave. After cooling, 0.1% of Na-ascorbate,dissolved in distilled water and filtered un der sterile conditionsthrough a Seitz filter, was added in order to reduce the Redoxpotential. The solution was then inoculated with spores of Clostridiumtyr0butyri= cum and incubated for 120 hours at 37 C. The spore formationwas then about The vegetative bacteria were then destroyed by heatingfor half an hour to 80 C.

e-Mainstage culture.Fermenters filled with 15 liters of the followingnutrient solution Percent Casein peptone 2 Meat extract 0.1 L-lysine0.05 KCl 0.25 FeSO 0.006

were inoculated with spores as described in Example 2. Then, 0.5% ofcane sugar, dissolved separately in distilled water and sterilized in anautoclave, and 0.05% of ascorbic acid, dissolved in distilled water andfiltered under sterile conditions through a bacteria filter, were added.After an incubation for 7 days at 37 C., of the bacteria had formedspores, so that the solution could be harvested.

(b) Recovery of the active substances 15 liters of Clostridia culturewere centrifuged and filtered. The clear filtrate was concentrated underreduced pressure to 500 cc. and dialyzed under toluene for 3 daysagainst flowing distilled water. The dialyzed culture solution was thenconcentrated under reduced pressure to 150 cc. and combined, whilestirring, with 750 cc. of ethanol. The precipitate was again dissolvedin 150 cc. of water, combined, while stirring, with 750 cc. of ethanoland centrifuged. Both alcohol solutions were combined and concentratedunder reduced pressure. The oil obtained thereby was taken up in waterand freezedried. Yield: 11.71g.

(c) Test for the bactericidal potency of the serum Upon administrationof gamma of the active substance, the Q -value after 16 hours amountedto 0.5 and after 24 hours, the Q -value was 0.5.

(d) Infection test mice were treated intraperitoneally with gamma of thesubstance obtained according to (b) in 0.5 cc. of water and after 24hours they were infected intravenously with Staphylococcus aureus. Thecontrol animals were given the same amount of physiological saltsolution.

Table III shows the survival rate of the treated animals in comparisonwith that of untreated control animals.

TABLE 3 [Survival rate of mice in the infection test] Number ofsurviving animals treated Untreated Days after with 2 gamma of controlinfection the substance obanimals tained according to (b) O 10 10 1 101O 2 10 7 3 9 7 4 8 7 5 8 ti 6 8 6 7 8 5 8 8 5 9 8 5 10 8 5 11 8 5 12 34 13 8 4 14 8 4 15 7 4 16 7 4 17 7 4 (e) Pyrogenicity The preparationobtained according to (b) is nonpyrogcnic in rabbits at 100 gamma/ kg.i.v.

(f) Toxicity A toxicity of the preparation could not befound. The LD inthe mouse is above 5 g./ kg.

EXAMPLE 4 (a) Cultivation of an apathogenic Clostridium straina-Prestage culture.Erlenmeyer flasks having a capacity of 500 cc. werecharged with a nutrient solution consisting of Percent Cane sugar 0.5Casein peptone 2 Meat extract 0.1

L-lysine 005 NaCl 0.25 pH-value=7.0.

and sterilized in an autoclave. After cooling, 0.1% of sodium ascorbate,dissolved in distilled water and filtered under sterile conditionsthrough a Seitz filter, was added in order to reduce the Redoxpotential. The solution was then inoculated with spores of Clostridiumacetobatyricum and incubated for 120 hours at 37 C. The extent of sporeformationwas then about 85%. The vegetative bacteria were then destroyedby heating'for half an hour to 80 C.

,B-Mainstage caltzt1'e.' Fermenters charged With 15 liters of thefollowing nutrient solution Percent Casein peptone 2 Meat extract 0.1

L-lysine 0.05 KCl 0.25 FeSO 0.006

were inoculated as described in Example 2 with the spores. Then, 0.5% ofcane sugar, separately dissolved in distilled water and sterilized in anautoclave, and 0.05% of ascorbic acid, dissolved in distilled water andfiltered under sterile conditions through a bacterial filter, wereadded. After incubation for 7 days at 37-C., of the-bacteria had formedspores, so that the solution could be harvested.

(b) Recovery of the active substances 15 liters of Clostridia culturewere centrifuged and filtered. The clear filtrate was concentrated underreduced pressure to about 500 cc. and then dialyzed for 3 days undertoluene against flowing distilled water. The dialyzed culture solutionwas then concentrated under reduced pressure to 150 cc. and combined,while stirring, with 750 cc. of ethanol. The precipitate was again dissolved in 150 cc. of water, combined while stirring with 750 cc. ofethanol and centrifuged. Both alcohol solutions were combined andconcentrated under reduced pressure. The oil was dissolved in 150 cc. ofWater and combined, while stirring, with 15 l. of acetone and thencentrifuged. The centrifuged precipitate was washed with ether and driedin an exsiccator. "Yield: 10.7 g.

(0) Test for the bactericidal potency of the serum After administrationof 5 gamma of active substance, the Q -value was 0.5 after 16 hours andafter 24 hours it was 0.5.

(d) Infection test 10 mice were treated intraperitoneally with 20 gammaof the substance obtained according to (b), dissolved in 0.5 cc. ofphysiological salt solution and after 24 hours they were infected byintravenous injection of Staphylococcus aureus. The control animals weregiven the same amount of physiological salt solution. I

Table 4 shows the survival rate of the treated animals in comparisonwith that of untreated control animals.

TAB LE 4 [Survival rate of mice in the infection test] N u'mber ofsurviving animals treated Untreated Days after with 20 gamma of controlinfection the substance obanimals tained according to (b) (e)Pyrogenicity The preparation obtained according to (b) is non- 75pyrogenic in rabbits at gamma/kg. i.v.

We claim:

1. A prOCesS for preparing alcohol-soluble high molecular weightmicrobial products which comprises preparing a sterile culture filtratecontaining said high molecular weight products from a culture of anapathogenic anaerobic Clostridium strain selected from the groupconsisting of Clostridz'um butyricum, Clostridium tyrobutyricum,Closlridium acetobutyriczrm, and Closlridium pectinovorum, and thenseparating said high molecular Weight products from the culturefiltrate.

2. A process as claimed in claim 1 wherein said Clost-ridium strain iscultivated for a period of 321 days at a temperature in the range of 20and 40 C., in a nutrient medium containing ascorbic acid.

3. A process as claimed in claim 1 wherein said Clost-ridium strain iscultivated for a period of 3-21 days at a temperature in the range of 20and 40 C., in a nutrient medium containing iron nails.

4. A process as claimed in claim 1 wherein said Clost-ridium strain iscultivated for a period of 321 days at a temperature in the range of 20and 40 C., in a nutrient medium through which nitrogen is passed.

5. The process as in claim 1 wherein said high molecular weightmicrobial products are separated from the culture filtrate by dialysis.

6. A process as claimed in claim 5, wherein the high molecular weightmicrobial products obtained from the culture filtrates by dialysis arefurther purified by adding to an aqueous solution of the said products alower aliphatic alcohol which is miscible with water, rejecting theprecipitate obtained and isolating the substances by concentration ofthe alcohol solution.

7. A process as claimed in claim 5, wherein the high molecular weightmicrobial products obtained by dialysis 10 from the culture filtratesare isolated from their aqueous solutions by precipitation with awater-soluble ketone.

8. The process as in claim 1 wherein said high molecular weightmicrobial products are separate-d from the culture filtrate by solventprecipitation.

9. A pharmaceutical preparation comprising an alcohol-soluble highmolecular weight microbial product prepared according to claim 1 inadmixture with a pharmaceutical carrier material.

10. A preparation as in claim 9 which is an injectable solution.

11. A preparation as in claim 9 which is an injectable aqueous solution.

12. A method for enhancing the unspecific resistance of an animalorganism which comprises administering to said organism an efiectiveamount of an alcohol-soluble high molecular weight microbial productprepared accordto claim 1 in admixture with a pharmaceutical carriermaterial.

References Cited by the Examiner UNITED STATES PATENTS 1/ 1964 Freedmanet al 167-78 FOREIGN PATENTS 886,597 11/1959 Great Britain.

OTHER REFERENCES Porter: Bacterial Chemistry and Physiology, publishedby John Wiley and Sons, Inc., New York, 1946, pp. 624, and 630-634.

A. LOUIS MONACELL, Primary Examiner.

D. M. STEPHENS, Assistant Examiner.

1. A PROCESS FOR PREPARING ALCOHOL-SOLUBLE HIGH MOLECULAR WEIGHTMICROBIAL PRODUCTS WHICH COMPRISES PREPARING A STERILE CULTURE FILRATECONTAINING SAID HIGH MOLECULAR WEIGHT PRODUCTS FROM A CULTURE OF ANAPATHOGENIC ANAEROBIC CLOSTRIDIUM STRAIN SELECTED FROM THE GROUPCONSISTING OF CLOSTRIDIUM BUTYRICUM, CLOSTRIDIUM TYROBUTYRICUM,CLOSTRIDIUM ACETOBUTYRICUM, AND CLOSTRIDIUM PECTINOVORUM, AND THENSEPARATING SAID HIGH MOLECULAR WEIGHT PRODUCTS FROM THE CULTUREFILTRATE.